The present invention is related to peptides coupled to charged polydispersive polysaccharides and their radiolabelled derivatives as well as their use in diagnostics and for treatment of cancer and other diseases.
Technetium-99m is provided with ideal physical properties as a radioactive marker in isotope-imaging. It is easily obtainable from a 99Mo/99mTc-generator, which is included in the isotope-laboratory equipment of each general hospital. The methods of labelling peptides with technetium-99m can be divided into two main categories, use of bifunctional metal chelates and use for labelling free thiol-groups. Rhenium, the isotopes 186Re and 188Re of which are suitable for radiotherapy due to their radiation properties, behaves chemically in the same way as technetium. In addition, many peptides have been labelled for diagnostic and therapeutic purposes with the radioactive isotopes of many other metals by using metal chelates. The most common metal chelates are derivatives of DTPA and EDTA. Further isotopes of halogens, especially iodine, have been used by coupling them to tyrosine and/or histidine residues possibly present in a peptide chain or to separate structures incorporated into the peptide chains.
Due to the general inhibitory effect of somatostatin, it has been tested in treatments of patients suffering from syndromes caused by neuroendocrine tumours. Neuroendocrine cancers are characterized by a large amount of somatostatin-receptors. A crucial problem in medical use of somatostatin is the fact that its biological half-life in blood serum is below three minutes. Due to this fact somatostatin analogs with longer in vivo half-lives have been developed in order to facilitate diagnoses and treatment of neuroendocrine tumours. So far the treatments with somatostatin analogs have mostly been limited to treating hormone-dependent symptoms of the patient.
Radiolabelled somatostatin derivatives are known for example from the following patents/patent applications: U.S. Pat. Nos. 5,225,180; 5,405,597; WO 92/21383 and WO 90/06949.
Successful diagnostic studies have been performed using somatostatin analogs 123I-Tyr3-octreotide (Lamberts et al., N. Eng. J. Med. 323: 1246-1249, 1990). However, said compound suffers from some disadvantages. It is secreted through the liver-kidney system and thus disturbs the imaging of the lower parts of the body. On the other hand, iodine-123 is an expensive isotope and the labelling technique is complicated.
A (111In-DPTA-Phel)octreotide, which is excreted from the body through the kidneys, thus facilitating the diagnoses of tumours inside the stomach without the accumulation of disturbing background-activity, has been developed from the above mentioned somatostatin derivative (Krenning E. P., et al., J. Nucl. Med. 33 :652-658, 1992). Another problem related to the octreotide is also the fact that it does not identify all types of somatostatin receptors. A disadvantage with the use of indium-111 is the fact that it is an expensive isotope and its availability is limited. Indium-111 has to be ordered by the hospital for each patient, separately. In addition, the radiation properties of indium-111 are not well adaptable to diagnostic and therapeutic use.
Somatostatin-dextran (Dextran 10) labelled with technetium-99m has a remarkably longer half-life in blood than native somatostatin(T(1)xc2xd=4 h and T(2)xc2xd=8 h) Holmberg, et al., Antibody, Immunoconj. Radiopharm 7: 253-259, 1994).
A further problem related to dextran somatostatin is non-specific binding to the cells, the transfer of the conjugate from the blood circulatory system to the lymphatic system and rather rapid excretion through the kidneys to urine.
Analogs of polysaccharide somatostatin with a negative effective surface charge are characterized by non-specific binding. When the transfer of the conjugate to the lymphatic system has been essentially decreased, it has been possible to regulate the transfer of the conjugate through the kidneys to the urine. It has been found that these compounds have special properties in treatment of cancer and they can be radiolabelled for in vivo diagnostics and therapy.
In the present invention the somatostatin analog has been coupled to a soluble polysaccharide with a negative effective surface charge. Hereinafter, said polysaccharides are referred to as charged polysaccharides. In addition, the charged polysaccharide somatostatins can be provided with groups, which in turn are able to bind detecting compounds, such as radionuclides, radiocontrasting substances or paramagnetic ions. Chelation or the production of another in vivo stable bond between the group and the detecting compound is used to carry out the coupling. Hereinafter said groups are referred to as chelates. If the chelate is able to bind only metals, the chelate is referred to as, a metal-chelate. A bond can be formed between the chelate and the detecting group before or after the chelate is coupled to the polysaccharide. It is also possible to couple some compounds directly to the structures of the polysaccharides, in which case a separate chelate is not required. Hereinafter, chelate is written within brackets in order to show that it is also possible to couple the detecting compounds directly to polysaccharides without a chelate. The accordingly formed charged polysaccharide-somatostatin-(chelate)-compounds are capable of binding to somatostatin receptors, which are expressed or over-expressed by tumours and metastases.
Said charged polysaccharide somatostatin (chelate) compounds are hereinafter referred to as the compounds according to the invention.
Compared to previously known methods, it was surprising that with a negative effective total charge a clear decrease in non-specific binding could be obtained and additionally by selecting a suitable polysaccharide size the half-life of the compound according to the invention in blood could be optimized.
A polysaccharide compound with high molecular-weight can be used when it is desired to maximize accumulation into the tumour, but the background concentration is without greater significance. On the contrary, when the background concentration is of importance, as in radiotherapy, the size of the polysaccharide is chosen in such a way that the radiation dose of the critical organ is not exceeded. In said case the size of polysaccharide is optimized in such a way that the accumulation kinetics in the tumour and the elimination of the compound according to the invention produces an optimal radiation dose relation between the tumour and the rest of the organism.
The object is the elimination of the compound according to the invention primarily through the kidneys, in which case the size of the compound should be less than 50 000 g/mol (grams/mole). In many diagnostic applications the background concentration should be low, too. Still, different methods have different clearance times.
One of the essential advantages of the method as compared to known techniques is the fact that it is possible to transport simultaneously to the target cells several somatostatins as well as several radioactive nuclides, if required.
All somatostatin analogs are not capable of identifying all types of somatostatin receptors. In the present invention such a somatostatin analog can be used, which identifies all types of somatostatin receptors or if required a somatostatin analog, which identifies only certain types of somatostatin receptors, thus targeting the compound to find its way to the target cell tissue. The biological half-life in blood is always remarkably dependent on the kind of charged polysaccharide to which the somatostatin analog is bound.
It is generally known that the charge of the polydisperse macromolecule is highly affected by the media in which the macromolecule of interest is situated. Said media dependent so called effective surface charge can deviate remarkably from the theoretical electric charge of the molecule based on the amount of dissociated groups. Said deviation is especially remarkable in a physiological medium, for example, in a human being, injected with said drug.
The evaluation of the effective surface charge is carried out with a multitude of different test systems, in which according to conventional methods based on electrophoresis, give the values, which best describe the actual situation. The effective surface charge can be determined exactly with test system based on convective electrophoresis. Effective surface charges of 10 charge units have been measured on macromolecules.
The polysaccharide can be straight-chained or branched. Preferably, the molecular-weight of the polysaccharide is 10 000-150 000 mol/g. One or more of the hydroxyl groups of the polysaccharide can be substituted independently by other functional groups; as non-limiting examples the following can be mentioned, xe2x80x94COOH, xe2x80x94NHCOCH3, NHSO3H, xe2x80x94OSO3H, xe2x80x94CH2OSO3H, xe2x80x94SO3H. When the radioactive label has been coupled to the polysaccharide it is preferred that the molecular-weight is 30 000-50 000 mol/g. When a non-labelled charged polysaccharide somatostatin compound is used it is more preferred that the molecular-weight of the polysaccharide is 50 000-80 00 mol/g.
Preferably, the polysaccharide is a polysaccharide formable by one or more different sugar units, with a mutual order which can be dependent or independent of the other. Preferred polysaccharides are dextrans.
In the compounds according to the invention the effective surface charge is obtainable by using compounds having a pKa of 7 or less. More preferred are compounds with a pKa of 4 or less. The following compounds, carboxylic acids and sulphonic acids, can be mentioned as non-limiting examples of such compounds. The charge has preferably been obtained by sulphonic acid groups. The effective surface charge can be 0.0005-1 unit charges per monomer. Preferred is an effective surface charge of 0.001-0.5 unit charges per monomer. Even more preferred is an effective surface charge of 0.002-0.2 unit charges per monomer.
In the compounds according to the present invention the chelate is bound by covalent bonds to the skeleton of charged polysaccharide. One or more chelates can be bound to the charged polysaccharide. Preferably, 0.005-0.5 chelates are bound to the charged polysaccharide per monomer. More preferably 0.05-0.3 chelates are bound to the charged polysaccharide per monomer.
The metal-ion can also be coupled directly to the polysaccharide skeleton. Such a coupling is preferred for technetium and rhenium.
The chelates can be coupled directly or they can be coupled through a bridge or through a mediating molecule to the charged polysaccharide or the somatostatin analog.
The term somatostatin analog includes somatostatins present in nature and analogs and derivatives thereof.
Derivatives and analogs have been used to mean any straight chained or straight-chained cyclic polypeptide derivative, present among somatostatins in nature, in which one or more unit has been removed or substituted by an amino acid radical and/or wherein one or more functional group has been substituted with one or more functional group and/or one or more group has been substituted with another isotheric group. Generally, the term covers all modified biologically active derivatives, which qualitatively fulfill an effect which is similar to that of unmodified somatostatin peptides. For example, they bind to somatostatin receptors or some subtypes of somatostatin receptors to decrease the secretion of hormones.
Cyclic, bridged-cyclic or straight-cyclic somatostatin analogs are known compounds. Such compounds and the preparation there of have been described for example in the European Patent Publications numbers EP 1 295; EP 29 579; EP 215 171; EP 203 031; EP 214 872; EP 298 732; EP 277 419.
Preferred compounds according to the invention are those charged polysaccharide somatostatins, in which the somatostatin analog is 
Most preferred compounds according to the invention are those in which the effective surface charge has been obtained with compounds, the structure of which have the formula II and
W1xe2x80x94(W2)nxe2x80x94Zxe2x80x83xe2x80x83II
in which:
W1 is a group, which can form an ether, ester or amino bond with a polysaccharide. W1 is preferably an amino group.
W2 is C1-6 alkyl, C1-6 arylalkyl or C1-6 alkyl, C1-6 arylalkyl, to the carbon atom of which an optional oxygen atom has been coupled or xe2x80x94NH or xe2x80x94SH or xe2x80x94COOH; -(W2)n can also be a combination of the groups mentioned above.
n is an integer from 0-7.
Z is COOH or SO3H.
Preferably, W1 is NH and W2 is CH2 and n is 1-3 and Z is SO3H.
Suitable chelate groups are physiologically acceptable chelates, which are capable of binding the detecting element. In addition, a preferred chelate group has a hydrophilic character. Examples of chelating groups are iminodicarboxylic groups, polyaminocarboxylic groups; the following are mentioned as examples of said non-cyclic ligand forming ethylendiaminetetraacetic acid (EDTA), triethylentriaminepentaacetic acid (DTPA), ethylenglycol-0,0xe2x80x2-bis(2-aminoethyl)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA), N,Nxe2x80x2-bis(hydroxibenzyl)ethylendiamin-N,Nxe2x80x2-diacetic acid (HBED), triethyltetraminehexaacetic acid (TTHA), derivatives thereof, in which one arm is stabilized as in cyclohexan-1,2-diamin-N,Nxe2x80x2,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-pentaacetic acid derivatives, derivatives of EDTA and DTPA as for example p-iso-thiocyanatobenzyl-EDTA or -DTPA, the macrocyclic derivatives thereof, for example 1,4,7,10-tetraazacyclodekan-N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x2xe2x80x3-tetraacetic acid (DOTA) and 1,4,8,11-tetraazacyclotetradekan-N,Nxe2x80x2,Nxe2x80x3N Nxe2x80x2xe2x80x3-tetraacetic acid (TETA), which are derivatized from N-substitutes or C-substituted macrocyclic amines including cyclamates as described e.g. in EP-A-304 780 and WO-A-89/01476, compounds the structures of which have the form III or IV 
in which
each R1, R2, and R3 are independently C1-6 alkyl, C6-8 aryl or C7-9 arylalkyl, additionally each can also be OH, C1-4 alkoxy, COOH or SO3H substituted
nxe2x80x2 is 1 or 2
i is an integer from 2-6 and
TT is independently of each other xcex1- or xcex2-amino acids coupled together with amine bonds.
As examples of chelating groups the following group, which has been Formed from bis-aminothiol derivatives can be mentioned, e.g. compounds with the formula V 
wherein
each R20, R21, R22 and R23 are independently of each others hydrogen or C1-4 alkyl,
X2 is an amino-, an acid group or a corresponding group, which is capable of reacting with a polysaccharide
mxe2x80x2 is two or three
compounds, which are derived from dithioazemcarbazone derivatives, e.g. compounds with the structure VI 
wherein
X2 is the same as above
compounds, which have been formed from amine-oxime derivatives, e.g. compounds having a structure with the formula VII 
wherein
R24, R25, R26, R27, R28 and R29 are independently H or C1-4 alkyl and X2 and mxe2x80x2 are as defined above,
compounds, which are diamidedimercaptide derivatives, e.g. compounds of the formula VIII 
wherein
X3 is a two-valanced radical, optionally substituted and comprising a group which is capable of reacting with a polysaccharide, e.g. C1-4 alkylene or phenyl including the group X2 and Y5 is hydrogen or CO2R30, wherein R30 is C1-4 alkyl
or compounds which are derivatives of porphyrins, such as N-benzyl-5,10,15,20-tetrakis- (4-carboxyphenyl)porphyrine or TPP comprising the group X2 as defined above.
Preferred chelate structures for halogens are compounds with the formula IX
W1xe2x80x94(W2)nxe2x80x94Arxe2x80x83xe2x80x83IX
wherein:
W1 is a group, which can form an ether-, ester- or amino-bond with a polysaccharide. Preferably W1 is an aminogroup.
W2 is C1-6 alkyl, C1-6 arylalkyl or C1-6 alkyl, C1-6 arylalkyl, to the carbon atom of which an optional oxygen atom has been coupled or xe2x80x94NH or xe2x80x94SH or xe2x80x94COOH; xe2x80x94(W2)n can also be a combination of the groups mentioned above.
n is an integer from 0-7.
Ar is an aryl group
It is advantageous if, when W1 is NH and W2 is CH2 n is 1-3 and Ar is benzyl
Preferably aryl means phenyl. Aralkyl preferably means benzyl.
Examples of X2 comprise radicals having the form xe2x80x94(X4)nxe2x80x3xe2x80x94X5 wherein X4 is C1-6 alkyl; or C1-6 alkyl to the carbon-atom of which optionally an oxygen atom has been coupled or xe2x80x94NHxe2x80x94, nxe2x80x3 is 0 or 1 and X5 is a group which can form an ether, ester or amino bond with a polysaccharide. It is to be understood that X2 is coupled to the carbon atom of xe2x80x94[CH2]nxe2x80x94 or xe2x95x90CHxe2x80x94CHxe2x95x90 by substituting a hydrogen atom.
The chelating group can be coupled directly or indirectly to the polysaccharide. When it is coupled indirectly it is preferably coupled through a bridge or intermediate structure, e.g. a group having the formula X
Zxe2x80x94R35xe2x80x94COxe2x80x94xe2x80x83xe2x80x83X
R35 is C1-11 alkylene, C2-11 alkenyl or xe2x80x94CH(Rxe2x80x2)xe2x80x94, wherein Rxe2x80x2 is a residue coupled to the xcex1-carbon in a native or synthetic xcex1-amino acid, e.g. hydrogen C1-11 alkyl, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl-methyl, Z is a functional group, which can react covalently with the chelate.
Z can or example be a group which can form an ether, ester or amine bond with the chelate. Preferably Z is an amino group.
The chelating group, when it is formed by carboxy, xe2x80x94SO3H and/or amino groups can be free or in the form of a salt.
Preferred chelating groups are those, which are formed from derivatives of polyaminopolycarboxylic groups, e.g. EDTA, DTPA, DOTA, TETA or substituted EDTA or DTPA.
In the compounds according to the invention the chelating group, when it is polyfunctional, can be coupled either to one polysaccharide molecule or to more polysaccharide molecules.
According to the invention the compounds can exist in free form or in the form of salts. Salts include salts formed from acid, e.g. organic acids, polymeric acids or inorganic acids, of which hydrochlorides and acetates are examples and forms of salts, which are formed from carboxylic groups or sulphonic acids of chelating groups, for example, alkali metal salts such as sodium or potassium or substituted or nonsubstituted ammonium salts.
The present invention is also related to a process for preparing the compounds according to the present invention. They can be prepared using analogs of known techniques.
Compounds according to the invention can be prepared for example in the following way: 
wherein Pa-OH is a polysaccharide unit and NH2xe2x80x94R represents 1) a somatostatin analog, 2) a compound with which a negative surface charge can be obtained as for example taurine 3) a chelate such as N-[2-amino-3(p-aminobenzyl)propyl]-cyclohexane-1,2-diamine-N,Nxe2x80x2,Nxe2x80x2,Nxe2x80x3-pentaacetic acid (CHXA-DTPA) and tyrosine.
In reaction phase 1 the polysaccharide is activated with NaIO4. In this case the cyclic polysaccharide ring structure is opened. In reaction phase 2 the desired amines and somatostatin are added, which react according to reaction formula 2 with the polysaccharide forming a Schiff bond. In the presence of the cyanoborohydride added in reaction phase 3 an amino bond is further created.
Alternative methods of synthesis have been described in the book Andreas Holmberg, Dextran conjugates for tumor targeting Synthesis and Characterisation, Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 546, ACTA Universitatis Upsaliensis, Uppsala 1995.
The compounds according to the invention can be purified with conventional methods, for example, with chromatography and ultrafiltration.
Preferably the compounds according to the invention comprise less than 5% of the weight of the peptide part of other groups (free chelates, unbound peptide, groups on which an effective surface charged has been obtained).
The compounds according to the invention in the basic form or in their pharmaceutically acceptable form are valuable compounds. As described below a detecting element can be coupled to the compounds according to the invention.